Genetic analysis of HA1 gene of influenza A (H3N2) viruses isolated from returning travelers at Chubu International Airport in Aichi Prefecture.

*Corresponding author: Mailing address: Department of Microbiology and Zoology, Aichi Prefectural Institute of Public Health, Nagare 7-6, Tsujimachi, Kita-ku, Nagoya 462-8576, Japan. Tel: +81-52-910-5674, Fax: +81-52-913-3641, E-mail: mami_hata@ pref.aichi.lg.jp Since the pandemic of 1968, the H3N2 subtype of influenza A virus (AH3) continues to circulate and has caused significant morbidity and mortality worldwide. Continuously accumulated mutations on the hemagglutinin (HA) gene of this virus have generated antigenically drifted strains that cause annual epidemics. Despite the recent dissemination of information regarding the evolution of circulating influenza viruses (1), questions remain about newly drifted viral emergence and how such viruses spread globally. However, it is generally accepted that frequent air travel plays an important role in the rapid and extensive spread of influenza (2). In Aichi Prefecture, we conducted a surveillance program to investigate influenza viruses carried by incoming travelers from abroad. The analysis of the influenza viruses isolated from travelers during the period of 1996 to 1999 revealed the possibility that viruses imported by air travelers may have influenced domestic influenza epidemics (3). In this report, we present a molecular epidemiological analysis of the HA1 (immunogenic subunit of HA) gene of the influenza viruses isolated from travelers during the period from 2006 to 2008, and the results were compared with those of analyses of domestic isolates obtained from residents of Aichi Prefecture. Throat-swab specimens were collected from travelers who reported flu-like symptoms to the Chubu International Airport Quarantine Branch Office (Tokoname, Japan). Specimens from domestic patients in Aichi Prefecture were also collected. All specimens were inoculated onto Madin-Darby canine kidney (MDCK) cells and the cultures were observed for characteristic cytopathic effects (CPEs) for up to 2 weeks. Serological types and subtypes were determined for each isolate by hemagglutination inhibition (HI) testing of CPEpositive MDCK culture supernatants using type-specific sera against influenza viruses (provided by the National Institute of Infectious Diseases, Japan). Viral RNA was extracted from the culture supernatants using the High Pure Viral RNA kit (Roche Applied Science, Penzberg, Germany). A region containing the complete HA1 gene was amplified from the viral RNA using the One-step RT-PCR kit (Invitrogen, Carlsbad, Calif., USA) and primers designed before (4). Sequencing of the amplified DNA fragments was carried out with a Model4200 automated DNA sequencer (Li-Cor, Lincoln, Nebr., USA). Fragments with a length of 987-nt of the AH3 virus HA1 gene were used for phylogenetic analysis by GENETYX (ver.7; Genetyx Corp., Tokyo, Japan). From the 26 specimens collected at the airport during two winter seasons (2004/2005 to 2006/2007), 11 influenza AH3 viruses and 2 influenza B viruses were isolated. The AH3 viruses were isolated from travelers who visited Singapore, Thailand, China, Hawaii, Italy, and Australia (Table 1). Among the regions visited, 6 (55%) samples were from people returning from Asia, and 3 (27%) were from people returning from Hawaii. We also analyzed 32 domestic strains of AH3 virus